Recombinant Pseudomonas putida D-tyrosyl-tRNA (Tyr) deacylase (dtd)

Basic Research Questions

• What is D-tyrosyl-tRNA (Tyr) deacylase (dtd) from Pseudomonas putida and what is its primary function?

  D-tyrosyl-tRNA (Tyr) deacylase (dtd) from Pseudomonas putida is an editing enzyme responsible for hydrolyzing ester bonds formed between D-amino acids and tRNA molecules. Its primary function is to prevent the incorporation of D-amino acids into proteins during translation by removing D-amino acids erroneously attached to tRNAs. The enzyme belongs to the α/β class of proteins and contains highly conserved active site motifs. The P. putida dtd is composed of 145 amino acids and has a Uniprot ID of Q88D02 .

  Recent research has demonstrated that dtd's function extends beyond the canonical "chiral proofreading" role. It also eliminates glycine molecules that have been mistakenly coupled to tRNA^Ala, indicating its broader role in translation quality control .

• How is the structure of Pseudomonas putida dtd organized and what are its key structural features?

  The structural architecture of P. putida dtd, like other bacterial DTDs, belongs to the α/β class of proteins. The structure typically contains:

  • A five-stranded mixed β-sheet and a three-stranded anti-parallel β-sheet

  • Two parallel α helices that cover the β-sheets

  • A highly conserved "SQFT" motif that forms the active site

  • An asymmetric unit consisting of a protein dimer

  This structural organization is highly conserved across species, with root mean square deviations of 1.1 to 1.9 Å when compared to DTDs from other organisms such as Haemophilus influenza, Aquifex aeolicus, Homo sapiens, Leishmania major, and Plasmodium falciparum .

• What experimental methods can be used to assess dtd enzyme activity in vitro?

  Several methodological approaches can be used to assess dtd enzyme activity in vitro:

  • Deacylation assays: Measuring the hydrolysis of D-amino acid-charged tRNAs by monitoring the release of free D-amino acids

  • Mass spectrometry: Confirming substrate specificity by analyzing the products of the enzymatic reaction

  • Fluorescence-based assays: Utilizing fluorescent-labeled substrates to monitor enzyme kinetics in real-time

  • Radiolabeled substrate assays: Using radiolabeled D-amino acids attached to tRNAs to quantify deacylation activity

  Specific activity can be measured by determining the amount of D-amino acid released from the charged tRNA over time under controlled conditions (pH, temperature, ionic strength) .

• How does dtd discriminate between D-amino acids and L-amino acids?

  The enantio-selectivity of dtd is achieved through its unique structural features:

  • The presence of a Gly-Cys-Pro dipeptide motif is responsible for maintaining homochirality by selecting only D-amino acids and rejecting L-amino acids

  • The enzyme contains specific subsites (transition site, active site, and exit site) that allow docking, re-orientation, chiral selection, catalysis, and exit of the free D-amino acid

  • The "Threonine" residue in the active site acts as the main nucleophile for catalysis, with phenylalanine and glutamine stabilizing the oxyanion hole during the cleavage of the ester bond

  Mutational studies involving the replacement of "Threonine" with "Alanine" have confirmed the role of threonine as the main active site residue essential for the enzyme's function .

Advanced Research Questions

• What is the mechanistic basis for dtd's ability to act on both D-amino acids and glycine attached to tRNAs?

  The dual functionality of dtd in hydrolyzing both D-amino acids and glycine from tRNAs is based on a sophisticated mechanistic foundation:

  • Glycine is an achiral amino acid, lacking a chiral center, which allows it to mimic the orientation of D-amino acids in the dtd active site

  • DTD's activity on Gly-tRNA^Ala is approximately 1000-fold higher than on Gly-tRNA^Gly, demonstrating a tRNA-based modulation of activity

  • The tRNA's discriminator base (N73) predominantly accounts for this activity difference, with uracil (U73) serving as a negative determinant that prevents Gly-tRNA^Gly misediting

  • The G3- U70 base pair, a universal tRNA^Ala-specific determinant, enhances DTD's activity by approximately 10-fold

  This enzymatic versatility allows dtd to serve as a crucial quality control factor in protein synthesis by eliminating both D-amino acids and misincorporated glycine .

• How does the discriminator base in tRNA affect dtd's activity in Pseudomonas putida?

  The discriminator base (N73) in tRNA plays a critical role in modulating dtd's activity:

  • Bacterial tRNA^Gly consistently has a pyrimidine (specifically uracil, U73) as its discriminator base, while most other tRNAs, including tRNA^Ala, have a purine

  • dtd exhibits approximately 100-fold higher activity on tRNAs with purine as N73 compared to those with U73

  • This discriminator base-dependent activity modulation prevents dtd from excessively hydrolyzing correctly charged Gly-tRNA^Gly (which has U73) while efficiently clearing misacylated Gly-tRNA^Ala (which has a purine at N73)

  The specific interaction between dtd and the discriminator base exemplifies an elegant evolutionary solution to the trade-off between speed and accuracy in translation quality control .

• How can recombinant P. putida dtd be optimally expressed and purified for structural and functional studies?

  For optimal expression and purification of recombinant P. putida dtd:

  Expression Systems:

  • E. coli-based expression systems using vectors with strong promoters (e.g., T7) yield good results

  • Baculovirus expression systems can be used for higher eukaryotic-like post-translational modifications

  Purification Strategy:

  1. Use affinity chromatography (His-tag or other fusion tags) for initial capture

  2. Apply size exclusion chromatography to obtain pure dimeric protein

  3. Ensure >85% purity by SDS-PAGE analysis

  Storage Conditions:

  • Store at -20°C/-80°C with 5-50% glycerol as a cryoprotectant

  • Avoid repeated freeze-thaw cycles

  • Working aliquots can be stored at 4°C for up to one week

  Reconstitution:

  • Reconstitute in deionized sterile water to a concentration of 0.1-1.0 mg/mL

  • Add glycerol to a final concentration of 50% for long-term storage

  The shelf life is typically 6 months at -20°C/-80°C for liquid preparations and 12 months for lyophilized forms .

• What approaches can be used to study protein-protein interactions involving dtd in Pseudomonas putida?

  Several approaches can be employed to study protein-protein interactions involving dtd in P. putida:

  1. Genetic code expansion with crosslinking amino acids:

     • Incorporate photocrosslinking amino acids like p-benzoyl-L-phenylalanine (pBpa) into dtd using the Mj TyrRS/tRNA system

     • UV-activate the crosslinker to capture interacting proteins in vivo

     • Analyze captured complexes by mass spectrometry

  2. Co-immunoprecipitation studies:

     • Express tagged versions of dtd in P. putida

     • Isolate protein complexes using antibodies against the tag

     • Identify interacting partners by mass spectrometry

  3. Yeast two-hybrid or bacterial two-hybrid screening:

     • Screen for potential protein partners using dtd as bait

     • Validate interactions using pulldown assays

  4. Fluorescence resonance energy transfer (FRET):

     • Tag dtd and potential interacting proteins with appropriate fluorophores

     • Monitor protein-protein interactions in real-time by measuring energy transfer

  Genetic code expansion in P. putida KT2440 has been established with 34.6-78% efficiency, making the incorporation of photocrosslinking amino acids a particularly promising approach for studying dtd interactions .

• What are the consequences of dtd overexpression or deletion in P. putida, and how can these phenotypes be characterized?

  Overexpression or deletion of dtd in P. putida leads to distinctive phenotypes that can be characterized through various experimental approaches:

  dtd Overexpression Consequences:

  • Toxicity due to depletion of cognate Gly-tRNA^Gly pool, as evidenced by northern blotting analysis

  • Complete depletion of aminoacylated tRNA^Gly, even with leaky expression

  • Potential growth defects due to disruption of protein synthesis

  dtd Deletion Consequences:

  • Increased sensitivity to D-amino acids

  • Potential accumulation of misfolded proteins due to D-amino acid incorporation

  • Possible growth defects in media containing D-amino acids

  Characterization Methods:

  1. Growth assays: Monitor growth curves in the presence of varying concentrations of D-amino acids

  2. Northern blotting: Analyze the aminoacylation status of tRNAs

  3. GFP reporter assays: Measure misincorporation rates using specially designed GFP constructs

  4. Proteomics: Identify proteins with altered abundance or D-amino acid incorporation

  5. Stress response analysis: Monitor expression of heat shock proteins and other stress markers

  These approaches can provide comprehensive insights into the physiological role of dtd in P. putida .

• How can P. putida dtd be engineered for novel functionalities in synthetic biology applications?

  Engineering P. putida dtd for novel functionalities can be approached through several strategies:

  1. Active site engineering:

     • Modify the "SQFT" motif to alter substrate specificity

     • Introduce mutations in residues that interact with the discriminator base to modify tRNA preferences

  2. Controlled expression systems:

     • Develop inducible systems for tight regulation of dtd expression

     • Create feedback-regulated dtd expression based on D-amino acid concentrations

  3. Novel applications:

     • Engineer dtd to selectively accept certain D-amino acids for incorporation into proteins

     • Create an in vivo system for generation of D-amino acid-containing peptides with therapeutic potential

     • Develop dtd variants that can remove non-canonical amino acids from tRNAs

  4. Domain fusions:

     • Create fusion proteins with other editing domains to expand functionality

     • Link dtd to biosensors for detecting D-amino acids in various environments

  These engineering approaches could facilitate the development of P. putida strains with enhanced capabilities for bioproduction, bioremediation, and other applications .

• What role does dtd play in the adaptive evolution of P. putida under stress conditions?

  During adaptive evolution under stress conditions, dtd may play significant roles in P. putida:

  1. Long-term stationary phase adaptation:

     • P. putida is known to adapt genetically during long-term stationary phase

     • Mutations may accumulate in dtd or its regulatory regions to optimize translation fidelity under stress

  2. Response to D-amino acid stress:

     • Increased expression of dtd may occur under conditions where D-amino acids are prevalent

     • This upregulation would protect against misincorporation during protein synthesis

  3. Contribution to chaotrope tolerance:

     • The expression of global regulators like PprI from Deinococcus radiodurans in P. putida enhances tolerance to various stressors

     • dtd may be part of the stress response network regulated by such global factors

  4. Metabolic adaptation:

     • The role of dtd in maintaining translation fidelity becomes critical during metabolic rewiring

     • In genome-reduced P. putida strains engineered for novel metabolic functions, proper dtd function ensures the correct production of engineered enzymes

  Understanding these adaptive roles can guide the development of more robust P. putida strains for biotechnological applications .

• How can dtd function be integrated into genome-scale metabolic models of P. putida?

  Integrating dtd function into genome-scale metabolic models of P. putida requires sophisticated computational approaches:

  1. Constraint-based reconstruction and analysis (COBRA):

     • Incorporate dtd-related reactions in existing models like iJN746

     • Define constraints based on kinetic parameters of dtd

  2. Multi-scale modeling approaches:

     • Link metabolic models with translation quality control processes

     • Include the energetic costs of proofreading and error correction

  3. Integration with omics data:

     • Use transcriptomic and proteomic data to refine dtd activity parameters in the model

     • Incorporate dtd regulation into gene regulatory network models

  4. Model validation:

     • Compare predicted phenotypes with experimental observations of dtd mutants

     • Use flux balance analysis (FBA) to predict the impact of dtd manipulation on growth and production

  5. Application to metabolic engineering:

     • Use the enhanced model to predict optimal dtd expression levels for specific bioprocesses

     • Identify potential interactions between dtd and engineered metabolic pathways

  Such integrated models can provide valuable insights for rational strain design in metabolic engineering applications of P. putida .